This invention relates to the quantitative analysis of human sputums for the presence of microbial pathogens. In another aspect, this invention relates to a novel technique of liquefying sputum specimens with a saponin mucolytic agent.
The condition of pneumonia in a human patient generally comprises an acute inflammatory condition of a lung or lungs which can be caused by microbial pathogens such as bacteria or viruses as well as chemical irritants or foreign bodies. In order to determine the causal agent in a patient with a presumptive diagnosis of pneumonia, samples of lung fluid are required. The ideal specimens of lung fluid are homogeneous samples which can be obtained by surgical intervention, for example, transtrachael aspiration. As is typical, surgical intervention techniques are time consuming and present a risk to the patient. Fatal reactions have been reported resulting from transtrachael aspiration. For example, hypotoxic patients and those suffering from debilitating diseases such as blood dyscrasia are especially prone to serious complications from transtrachael aspiration.
As a consequence, most physicians rely on the early morning couph sputum specimen as the means of obtaining a lung fluid specimen. This technique is simple and very common. Unfortunately, the cough sputum specimen can be readily contaminated by the normal flora in the mouth, nose, posterior pharynx, and stomach. Furthermore, sputums from patients with pneumonia are very viscous and heterogeneous in nature and therefore difficult to disperse in a reproducible manner. Quantitative analysis of sputum cannot be accomplished on the very viscous heterogeneous sputums because it is generally necessary in quantitative analysis of specimens like sputum to dilute the specimen and thereafter plate minor portions of the specimen on various growth media and then determine the type and number of colonies of microbial organisms which result on the media. Generally, the maximum number of colonies which can be effectively counted on a petri dish is about 300. Furthermore, it has been accepted that normal sputum samples can contain contaminating microorganisms in quantites up to 10.sup.5 per milliliter, whereas, as causative organisms of pneumonia microorganisms are generally present in quantities greater than about 10.sup.6 per milliliter. Therefore, before a sputum specimen can be quantitatively analyzed, it must be diluted in a manner such that the microbial pathogens are uniformly dispersed in the resulting diluted sample. Accordingly, if a minor portion of this diluted sputum specimen is quantitatively analyzed for the type and number of microbial pathogens, the accurate number of microbial pathogens per milliliter of the sputum sample can be accurately calculated.
Thus, in order to reduce the problem of external contamination and heterogeneity of the sputum sample and provide a quantitative sputum analysis, several attempts have been made to digest the sputum specimen with enzymatic and chemical digestants. Several such digestants have been theretofore tried and all of the digestants have one or more of the following disadvantages: expensive; short shelf life; temperature sensitive; toxic to some or many pathogenic organisms; and require long digestion time. For example, a number of proteolytic enzymes have been tested in both purulent and mucoid sputum. Of such enzymatic materials, trypsin, elastase, and chymotrypsin appear the most effective, and enzymes such as bromelain, ficin or papain were only effective at extreme high concentrations, while plasmin has no detectable effect on sputum viscosity. All such proteolytic enzymes appear to be more effective with mucoid sputums than with purulent sputums. The most widely used digestants for quantitative sputum analysis are aqueous solutions of N-acetylcysteine and Cleland's reagent (1,4-dithio-mesoerithritol). In general, Cleland's reagent exhibits greater mucolytic activity than N-acetylcysteine at lower concentrations, but Cleland's reagent generally loses its mucolytic activity in relatively short periods of time in aqueous solution. Furthermore, both of these reagents are somewhat toxic to microbial pathogens at concentrations needed for rapid digestion of sputum.
Consequently most sputum specimens are processed by a nonquantitative streaking technique, which generally involves streaking the heterogeneous sputum specimen on various growth media. These techniques lead to a substantial number of false positive cultures, and in many instances, overgrowth of the pathogenic organisms by contaminating microorganisms.